Project Summary Failures of antibiotic therapy occur with increasing frequency in clinics due to the spread of multidrug resistant bacterial pathogens. The major challenge are infections caused by Gram-negative bacteria that are protected from antibiotics by the concerted action of multidrug efflux pumps and the low permeability barrier of outer membranes. Pseudomonas aeruginosa, an opportunistic human pathogen responsible for a variety of infectious diseases, is notorious for its antibiotic impermeability. A handful of clinical antibiotics used against this pathogen are beginning to fail due to the emergence of multidrug resistant strains. New antibiotics are needed to address this growing threat. Decades of antibiotic discovery and optimization perfected empirical approaches for improvement of antibiotic action and avoidance of class-specific resistance mechanisms. The current critical challenge is to develop approaches that will enable antibiotic penetration across non-specific permeability barriers of Gram-negative bacteria, which present an urgent and serious threat to public health. This project responds to this challenge and proposes the development of a new technology for optimization of efflux avoidance and inhibition in clinical and investigational antibacterial agents that will be effective against Gram-negative bacteria. The proposed approach targets simultaneously the multidrug efflux mechanism of P. aeruginosa and its outer membrane barrier and combines cutting edge technologies in experimental analyses of efflux inhibition and drug penetration, kinetic modeling of drug accumulation, computer simulations of drug efflux and transmembrane diffusion, synthetic chemistry and machine learning analyses. The central objective of the proposal is to create a mechanism-based predictive model that integrates physicochemical properties of compounds, kinetics of their intracellular accumulation and transmembrane diffusion, and a molecular level description of the interaction of efflux transporters with their substrates and inhibitors. The model will be validated by focused medicinal chemistry efforts to generate antibacterial agents that combine the traits of effective antibiotics and potent efflux pump inhibitors or avoiders. This multi-disciplinary approach is enabled by the collaborative efforts of PIs on the project: Helen Zgurskaya (biochemistry of drug uptake and efflux), Valentin Rybenkov (kinetic modeling), Paolo Ruggerone (computational biophysics of efflux), Gnanakaran (computational simulations of membrane permeation) and John Walker (medicinal chemistry).